Vaccine compositions and adjuvant

ABSTRACT

The immune response of an animal to a target immunogen may be enhanced by use of a novel adjuvant which includes low concentrations of killed cells of  Mycobacterium avium  subspecies  avium  in combination with mineral oil. The adjuvant may be used in vaccine compositions for the immunization of an animal against any target immunogen, and is particularly preferred for use with immunocontraceptive vaccines such as GnRH and PZP immunocontraceptive vaccines.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to novel vaccine compositions, includingimmunocontraceptive vaccines, and particularly to novel adjuvants foruse therein.

2. Description of the Prior Art

Gonadotropin releasing hormone (“GnRH”, also known as LuteinizingHormone Releasing Hormone, or “LHRH”), has long been recognized as beingof central importance to the regulation of fertility in animals. GnRH isa decapeptide which has the same amino acid sequence, i.e.,pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH₂ (SEQ. ID NO. 1) in allmamals. Closely relate GnRH compounds have also been identified in othernon-mammals, including fowl, and receptors for GnRH have been identifiedin reptiles and amphibians. In males and females, GnRH is released fromthe hypothalamus into the bloodstream and travels via the blood to thepituitary, where it induces the release of the gonadotropins,luteinizing hormone (LH) and follicle stimulating hormone (FSH). Thesetwo gonadotropins, in turn, act upon the gonads, inducingsteroidogenesis and gametogenesis. In growing male animals, thegonadotropins stimulate the development of the testes and the synthesisof testicular steroids. In the growing female animal, the development ofthe ovaries is stimulated and therein follicle development, synthesis ofovarian steroids and ovulation. Steroids released from the gonads intothe circulation also act upon various other tissues.

In recent years, GnRH neutralization has been used as an effective meansof contraception in a variety of animals. Fraser described that thegonadotropin hormonal cascade can be halted by neutralization of thebiological activity of GnRH (Physiological Effects of Antibody toLuteinizing Hormone Releasing Hormone. In: Physiological Effects ofImmunity Against Reproductive Hormones, Edwards and Johnson, Eds.Cambridge University Press, 1976). As a consequence of GnRHneutralization, the gonadotropins and gonadal steroids are not releasedinto the blood, interrupting the hormonal regulation of fertility andceasing gametogenesis. In addition to the use of immunization againstGnRH for animal sterilization to prevent breeding, the immunization hasalso been suggested for the treatment of aggressiveness in male animalssuch as dogs and bulls, chemical castration of male animals forslaughter, prevention of heat in female animals, and prevention ofrestlessness in male animals being fattened for slaughter, and reductionof boar taint in the meat of pigs raised for slaughter.

Neutralization of GnRH has also been employed for the treatment of anumber of other diseases. A number of important diseases, includingbreast cancer, uterine and other gynecological cancers, endometriosis,uterine fibroids, prostate cancer, and benign prostatic hypertrophy, arealso affected by gonadotropins and gonadal steroid hormones.Neutralization of the patient's GnRH effectively eliminates the gonadalsteroids that induce and stimulate these diseases. See McLachlan et al.,1986, British Journal of Obstetrics and Gynecology, 93:431-454; Conn andCrowley, 1991, New England Journal of Medicine, 324:93-103; Filicori andFlamigni, 1988, Drugs, 35:63-82.

GnRH neutralization has been typically achieved by the induction orintroduction of anti-GnRH antibodies in the subject animal or patient.These antibodies may be induced by active immunization with GnRHimmunogens, or by passive immunization by administering anti-GnRHantibodies (Fraser, 1976, ibid). Antibodies to GnRH produce infertilityby binding to circulating endogenous GnRH, precluding the GnRH frombinding to its pituitary receptor and thereby interfering with itsability to release FSH and LH. The severe reduction or absence of thesehormones leads to atrophy of the gonads and concomitant infertility inboth sexes as described above.

Despite these advantages, active immunization against GnRH has not beenwidely practiced due to deficiencies associated with the GnRH vaccines.The prior art anti-GnRH vaccines have typically lacked the potencynecessary to effect long-term contraception in a single dose. In fact,immunocontraception has traditionally required at least two doses, aprime and a boost, for long-term efficacy. The prime dose prepares theimmune system for repeat antigen exposure and provides only a short termresponse. The subsequent boost immunization can result in an immuneresponse which can be maintained for a period of months to years. Inaddition to the GnRH immunogen, an adjuvant is a necessary component ofany vaccine intended for long-lasting immunocontraception.

At present, Freund's complete adjuvant (FCA) is the only adjuvant thathas provided high and long-lasting immunocontraceptive responses.Although many other adjuvants have been developed, none have been ableto achieve the high antibody titers obtained using Freund's completeadjuvant. Freund's complete adjuvant includes a emulsion of killedbacteria of Mycobacterium tuberculosis or M. butyricum (also known as M.smegmatis) in mineral oil with a surfactant.

Despite the efficacy achieved with Freund's complete adjuvant, numerousconcerns have been raised over its use in animals, and particularly inanimals raised for human consumption. One primary concern has been thepotential for false-positive TB skin tests in an animal which has beeninjected with FCA containing killed M. tuberculosis (Tizard, 1977, AnIntroduction to Veterinary Immunology, CRC Press, Boca Raton, Fla.).Other concerns over the use of FCA have included fears that it may becarcinogenic and that it may cause intense cell-mediated immuneresponses which produce lesions at the site of injection.

SUMMARY OF THE INVENTION

We have now invented improved methods and compositions for vaccinatinganimals. In accordance with this invention, the immune response of ananimal to a target immunogen may be enhanced by use of a novel adjuvantwhich includes low concentrations of killed cells of Mycobacterium aviumsubspecies avium in combination with mineral oil. While the adjuvant maybe used in vaccine compositions for the immunization of an animalagainst any target immunogen, it is particularly preferred for use withimmunocontraceptive vaccines such as gonadotropin releasing hormone(GnRH) and porcine zona pellucida (PZP) immunocontraceptive vaccines.

In accordance with this discovery, it is an object of this invention toprovide vaccine compositions having a novel adjuvant.

Another object of this invention is to provide a novel adjuvant for usewith vaccine compositions which provides superior enhancement of immuneresponse to the target immunogen but which produces substantially noinflammation at the site of injection.

Still another object of this invention is to provide novel vaccinecompositions for the immunocontraception of animals.

Yet another object of this invention is to provide novel contraceptivevaccine compositions which are effective for long periods of time withonly a single shot.

Other objects and advantages of this invention will become readilyapparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the first study of Example 3 over fourconsecutive seasons with deer immunized with a prime dose followed by asecond, boost dose.

FIG. 2 shows the results of the second study of Example 3 over twoseasons with female deer using only a single shot of the vaccine.

FIG. 3 shows the results of the study of Example 4 with female pigs.

FIGS. 4(a) and 4(b) shows the results of the study of Example 4 withfemale pigs.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this invention, we have developed novel adjuvants foruse in the active immunization of animals. Traditionally, vaccines areprepared using a combination of an immunologically effective amount ofan immunogen of interest together with an adjuvant effective forenhancing the immune response of the animal against the immunogen. Wehave unexpectedly discovered that compositions of mineral oil with lowconcentrations of killed cells of Mycobacterium avium subspecies aviumprovide effective enhancement of immune responses and thus are effectivefor use as adjuvants. Moreover, this adjuvant provides enhanced immuneresponses which exhibit high, long-lasting antibody titers to theimmunogen, even after administration of only a single dose of thevaccine.

As used herein Mycobacterium avium subspecies avium refers to therecognized species M. avium subspecies avium, the characteristics ofwhich are described by Thorel et al. (1990, International Journal ofSystematic Bacteriology, 40:254-260, the contents of which areincorporated by reference herein) and the type strain of which has beendeposited at the American Type Culture Collection, Manassas, Va., USA,as deposit accession number ATCC 25291. In contrast, as used hereinMycobacterium avium does not refer to the M. avium “complex” which hasbeen used in the art to collectively include both of the two subspeciesM. avium subsp. avium and M. avium subsp. paratuberculosis.

Without wishing to be bound by theory, it is believed that the highefficacy of the M. avium subsp. avium containing adjuvant is due to thenearly ubiquitous presence of this microorganism in nature. Indeed, mostliving animals, including humans, have been exposed to M. avium subsp.avium in the environment. We believe that because most animals have beennaturally exposed to the microorganism, when they are initially injectedwith the immunogen plus adjuvant the immune response is enhanced by aspecific response to the M. avium subsp. avium which is similar to thatof a booster injection. The initial injection with the adjuvant of thisinvention therefore elicits an immune response which is usually seenonly after a boost injection of other adjuvants.

The particular strain of M. avium subsp. avium used in the preparationof the adjuvant is not critical. M. avium subsp. avium suitable for usein the adjuvant may be obtained from a variety of sources includingknown substantially pure strains or it may be isolated from naturalsources such as fowl or other animals using conventional techniques. Forcommercial production of the adjuvant, large quantities of cells of themicroorganism are preferably prepared by culture of the selected strain.Alternatively, killed cells may be obtained directly from commercialsources, such as the Johne's disease vaccine, MYCOPAR (manufactured byFort Dodge Animal Health, Overland Park, Kans., USA, and distributed bySolvay Animal Health, Mendota Heights, Minn., USA), which consists ofkilled cells M. avium subsp. avium strain 18.

Propagation of the microorganism for preparation of the adjuvant may beaccomplished by culture under any conventional conditions and on mediawhich promote its growth. Although a variety of conventional solid andliquid media may be suitable for use herein, growth in liquid culture isparticularly preferred for large scale production. Without being limitedthereto, Middlebrook broth is preferred. The microorganism will growover wide temperature ranges, although growth between 34°-38° C. istypically preferred. Once a sufficiently heavy growth of themicroorganism has been obtained, usually in about 10-25 days, the cellsmay be recovered and separated from the culture medium using techniquesconventional in the art, such as by centrifugation or filtration.Following separation, the cells may be further washed to removecontamination by extraneous microbial products or culture mediacomponents.

Following their propagation and recovery, cells of M. avium subsp. aviumare subjected to chemical and/or physical treatment effective to kill(i.e., inactivate) the cells. An effective treatment for killing thecells is defined herein as that which kills 99.9% or more of the viablecells, without lysing the cells and while retaining the ability of thecells to elicit an antibody response in the animal. Thus, the treatmentshould not substantially alter the specificity of the cell surfaceantigens on the killed cells relative to the untreated cells. Whiletreatments killing 100% of all viable cells would typically bepreferred, particularly for any applications involving treatment ofhumans, the skilled practitioner will recognize 100% cell death may notalways be readily obtainable. In the preferred embodiment, killed,intact M. avium subsp. avium are prepared by treatment of the viablecells with alcohol, particularly an aliphatic alcohol such as ethanol orisopropyl alcohol. Alternatively, the cells may be killed by UVirradiation such as described by Purdy et al. (U.S. Pat. No. 6,303,130)for the preparation of Pasteurella haemolytica bacterins. It is alsoenvisioned that a variety of other techniques have been described forthe preparation of killed cell vaccines (i.e., bacterins) are alsosuitable for use herein, and include but are not limited to treatmentwith phenol, tricresol, formalin, formaldehyde, acetone, merthiolate,and moderate heat at temperatures which would not induce proteindenaturation (e.g., 56° C. for 1 hour). Treatment times and conditionswill of course vary with the particular method selected and may bereadily determined by routine testing.

Adjuvant formulations are prepared by combining the killed cells withmineral oil, which is also used in the preparation of the well-knownFreund's adjuvant, and an optional surfactant or emulsifier. Inclusionof a surfactant is indicated when either or both of the M. avium subsp.avium killed cells or the immunogen of interest are in an aqueoussolution or suspension. The particular surfactant used is not critical,and a variety of surfactants are suitable for use herein for emulsifyingany aqueous components. Although mannide monooleate is a generallypreferred surfactant, examples of alternative surfactants which may alsobe used include but are not limited to polyoxyethylene ethers (oroctoxynols) such as lauryl, cetyl, oleyl, stearyl, and tridecylpolyoxyethylene ethers; polyoxyethylene sorbitan-fatty acid esters(commonly sold under the trade name TWEEN by ICI Americas Incorporated,Wilmington, Del.), such as polyoxyethylene(20)sorbitan monolaurate(TWEEN 20), polyoxyethylene(60)sorbitan monolaurate (TWEEN 60);polyoxyethylene ethers such as TRITON X-100, X-102, X-165, and X-305;fatty acid diethanolamides such as isostearic acid DEA, lauric acid DEA,capric acid DEA, linoleic acid DEA, myristic acid DEA, oleic acid DEA,and stearic acid DEA; fatty acid monoethanolamides such as coconut fattyacid monoethanolamide; fatty acid monisopropanolamides such as oleicacid monoisopropanolamide and lauric acid monoisopropanolamide; alkylamine oxides such as N-cocodimethylamine oxide, N-lauryl dimethylamineoxide, N-myristyl dimethylamine oxide, and N-stearyl dimethylamineoxide; N-acyl amine oxides such as N-cocoamidopropyl dimethylamine oxideand N-tallowamidopropyl dimethylamine oxide; and N-alkoxyalkyl amineoxides such as bis(2-hydroxyethyl) C₁₂-C₁₅ alkoxy-propylamine oxide. Theamount of the surfactant, if used, is not critical but should besufficient to emulsify any aqueous components. Consequently, therelative amounts of mineral oil to surfactant in the adjuvant willtypically be between about 85:15 and about 100:0, by weight,respectively. In the preferred embodiment, the ratio of mineral oil tosurfactant may vary between about 85:15 to 95:5, respectively, and inparticularly preferred embodiment is about 95:5.

In contrast to the mineral oil and surfactant, the amount of killedcells of M. avium subsp. avium is critical. The objective of theadjuvant of this invention coincides with the well-established use ofadjuvants in the active immunization art, which is to enhance the immuneresponse of an animal to immunization with a target immunogen,specifically, to enhance the production of antibodies against the targetimmunogen. Thus, the adjuvant is administered in a vaccine compositionwhich includes a target immunogen of interest, wherein the targetimmunogen is itself present in an immunologically effective amount. Asused throughout the art and herein, an immunologically effective amountof the target immunogen is defined as that amount which will elicitproduction of antibody by the subject animal against the immunogen.

Consequently, in accordance with the instant invention, the absoluteamount of the killed cells of M. avium subsp. avium and theirconcentration in the final vaccine composition (which includes thetarget immunogen) which is administered to the subject animal areselected to provide an effective enhancement (i.e., increase) of theproduction the antibody against the target immunogen as compared to acontrol animal (treated with a vaccine composition lacking theadjuvant). However, the amount of the killed cells of M. avium subsp.avium in the vaccine composition should not be so high that it wouldelicit a substantial T cell-mediated delayed hypersensitivity response(i.e., a Type IV response) by the animal to the M. avium subsp. avium ifthe adjuvant were administered alone without immunogen. A substantial Tcell-mediated delayed hypersensitivity response to M. avium subsp. aviumis defined herein as a skin reaction at the site of injection of thevaccine which is visible to the naked eye. Although the administrationof the inventive adjuvant may produce a reaction on a microscopic levelwhich may be seen upon microscopic examination of biopsy material, nogranuloma at the site of injection will be visible to the naked eye.Thus, the amount of the killed cells used in the vaccine will be muchless than that which might be typically used for a target immunogen. Theprecise effective amount of the killed cells used in the vaccinecomposition may vary somewhat with the particular target animal and itssize, and the stage of the vaccination (initial or single dose, orsecond or boost dose) and may be determined by the practitioner in theart by routine experimentation. Without being limited thereto, theconcentration of said killed cells of M. avium subsp. avium in thevaccine formulation administered to a subject animal will typically varybetween about 50 μg per ml and about 400 μg per ml, measured as the dryweight of said killed cells per ml of the vaccine composition. Withinthis range, the initial dose of vaccine formulations administered to ananimal in either a single or multiple dose program will generally have agreater amount of killed cells of M. avium subsp. avium, than boostdoses. Further, as a practical matter, the volume of vaccinecompositions which may be administered to animals parenterally byinjection is relatively small, no greater than about 1 ml for all butvery large animals such as elephants or whales. Consequently, the volumeof adjuvant and hence the amount of killed cells of M. avium subspeciesavium in the vaccine composition is also limited. Thus, in a preferredembodiment for the treatment of animals weighing less than about 2,000pounds (i.e. animals except the above-mentioned very large animals), thevaccine composition will typically contain more than or equal to about50 μg and less than or equal to about 400 μg of killed cells of M. aviumsubsp. avium by weight, and particularly more than or equal to about 50μg and less than or equal to about 200 μg of the killed cells by weight.

The manner of formulating the adjuvant and immunogen containingpreparation may vary with the phase of the immunogen preparation, theconcentration of the immunogen in the preparation, its solubility, andthe carrier used for the immunogen preparation, if present. Forinstance, without being limited thereto, when formulated with aqueousphase solutions or suspensions of an immunogen, the adjuvant andimmunogen preparation are preferably formulated in approximately equalvolumes, vigorously agitated to form an emulsion, and finally stiffenedby passage through a needle as is conventional in the art. Conversely,lyophilized immunogen preparations may be mixed directly with theadjuvant without the need for emulsification. Immunogen preparations inoil miscible carriers may also be simply mixed with adjuvant, againpreferably in approximately equal volumes.

The M. avium subsp. avium containing adjuvants of the invention may beused in combination with a variety of known immunogens or antigens usedfor active immunization of animals by parenteral injection to elicitproduction of antibodies reactive with the immunogen. The adjuvants arepreferred for use with GnRH, and particularly with GnRH or porcine zonapellucida (PZP) immunocontraceptive vaccines. However, it is alsoenvisioned that the adjuvants may be used with virtually any other knownimmunogen of interest other than M. avium (such as Johne's diseasevaccine). Thus, the adjuvant may be used with pathogenic microorganisms(living, attenuated, or killed) or biological molecules includingtoxoids, polysaccharides, proteins, peptides, or microbial subunits,which further include relatively large molecules which are themselvesantigenic in a target animal as well as smaller haptens or selfmolecules conjugated to immunogenic carriers. Examples of immunogensused in vaccination programs which may be used with the adjuvants of theinvention include but are not limited to Pasteurella haemolytica, Vibriocholera, Corynebacterium diptheriae toxoid, Hepatitis B viral antigen,Influenza virus, Measles virus, Meningococcal polysaccharide, Mumpsvirus, killed cells of Bordetella pertusis, Streptococcus pneumoniae(pneumococcus) polysaccharide, Polio viruses, Rabies virus, Rubellavirus, poxviruses such as Vaccinia virus, Clostridium tetani toxoid,Mycobacterium bovis, killed cells of Salmonella typhi, and Yellow fevervirus.

In a particularly preferred embodiment, the adjuvant is formulated witha GnRH or GnRH immunogenic mimic containing vaccine for inducingproduction of anti-GnRH antibody in an animal and thereby effecting oneor more responses ranging from contraception in males and/or females,reducing aggressive behavior in male animals, chemical castration,control or prevention of estrus or heat, prevention of restlessness inanimals prior to slaughter, reduction of boar taint in the meat of pigsraised for slaughter, and treatment of diseases as is known in the art.

While GnRH may be used in the immunogen preparation, a variety of GnRHimmunogenic analogs have also been described which are suitable for useherein. As noted hereinabove, GnRH is a small decapeptide having theamino acid sequence: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (SEQ.ID NO. 1). Throughout this description, the amino acid sequences conformwith conventional practice with the amino terminal amino acid on theleft and the carboxy terminal amino acid to the right. As definedherein, immunogenic analogs of GnRH include compounds containing asubstitution, deletion, or insertion of between one and five amino acidresidues in the above-mentioned GnRH amino acid sequence, as well asdimers or polymers thereof, which compound retains the ability to induceor stimulate the production in a subject animal of antibodies which bind(i.e., cross-react) to GnRH. The GnRH analog will preferably retain atleast five consecutive amino acids from the GnRH decapeptide. Thesubstitutions and insertions can be accomplished with natural ornon-natural amino acids, and substitutions are preferably conservativesubstitutions made with amino acids which maintain substantially thesame charge and hydrophobicity as the original amino acid. Moreover, theanalog may itself be immunogenic or it may be coupled to an immunogeniccarrier such as described hereinbelow.

Immunogenic analogs of GnRH which are suitable for use herein have beendescribed, for example, in Meleon (U.S. Pat. Nos. 5,484,592 and6,284,733), Mia (U.S. Pat. No. 4,608,251), Ladd et al. (U.S. Pat. No.5,759,551), Hoskinson et al. (published PCT application WO8805308), andRussell-Jones et al. (U.S. Pat. No. 5,403,586) the contents of each ofwhich are incorporated by reference herein. Thus, suitable GnRH analogsinclude but are not limited to GnRH peptides wherein the Gly at position6 of the GnRH decapeptide has been replaced by a dextrorotary (D)-aminoacid such as D-trp, D-glu, or D-lys (SEQ. ID NO. 2, 3, and 4,respectively); GnRH peptides wherein the p-Glu at position 1 of the GnRHdecapeptide has been replaced by a Glu, His, or Pro (SEQ. ID NO. 5, 6,and 7, respectively); any continuous 5, 6, 7, 8, or 9 amino acidfragment of the GnRH decapeptide, such as pGlu-His-Trp-Ser-.Tyr,pGlu-His-Trp-Ser-Tyr-Gly, pGlu-His-Trp-Ser-Tyr-Gly-Leu,His-Trp-Ser-Tyr-Gly-Leu-Arg, Trp-Ser-Tyr-Gly-Leu-Arg,Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, and Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (SEQ.ID NOS. 8-14, respectively); naturally occurring chicken GnRH II,pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH₂ (SEQ. ID NO. 15); naturallyoccurring salmon GnRH, pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH₂(SEQ. ID NO. 16); the nona- or decapeptide(Cys)-Lys-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, wherein the aminoterminal Cys is optional (SEQ. ID NOS. 17 and 18, respectively) or adimer of the decapeptide wherein the amino terminal Cys are coupled toone another (SEQ. ID NO. 19); a polymer of two or more decapeptides intandem of the formulaZ¹-Glx-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro[-Gly-X-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro]_(n)-Gly-Z²wherein n is an integer greater than or equal to 1, X is a direct bondor a spacer, Z¹-Glx is pGlu or Gln having an amino acid tail dttachedthereto for coupling to a carrier protein, and Gly-Z² is Gly-NH₂ or Glyhaving an amino acid tail attached thereto for coupling to a carrierprotein (SEQ. ID NO. 20); and a peptide having the sequencepGlu-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Gly-Gln-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Gly-Cyswherein Y is independently Gly or a D-amino acid which may optionallycontain an amino acid side chain attached thereto for coupling to acarrier protein (SEQ. ID NO. 21) or a dimer thereof.

While the GnRH may be isolated from natural sources, for practicalpurposes GnRH or and its analogs may be synthesized by a variety ofconventional methods Such techniques include but are not limited tomethods well known to those skilled in the art of peptide synthesis,e.g., solution phase synthesis [see Finn and Hoffman, In “Proteins,”Vol. 2, 3rd Ed., H. Neurath and R. L. Hill (eds.), Academic Press, NewYork, pp. 105-253 (1976)], or solid phase synthesis [see Barany andMerrifield, In “The Peptides,” Vol. 2, E. Gross and J. Meienhofer(eds.), Academic Press, New York, pp. 3-284 (1979)], or stepwise solidphase synthesis as reported by Merrifield [J. Am. Chem. Soc. 85:2149-2154 (1963)], the contents of each of which are incorporated hereinby reference.

Because GnRH is a small, self-molecule it should be conjugated directlyor indirectly to an immunogenic carrier in order to increase the immuneresponse to the peptide. A plurality carriers and carrier couplingtechniques have been previously described for GnRH or its analogs andare also suitable for use herein. See for example, Meleon, Mia, Ladd etal., Hoskinson et al., and Russell-Jones et al. mentioned above.However, in a preferred embodiment, GnRH or an analog thereof isconjugated to immunogenic mollusk hemocyanin carrier protein, directlyor indirectly through the C-terminal end of the GnRH or analog. Suitableimmunogenic mollusk hemocyanin proteins include Concholepas concholepashemocyanin protein, Keyhole Limpet (Megathura crenulate) hemocyaninprotein (KLH), Horseshoe crab (Limulus polyphemus) hemocyanin protein,and Abalone (Haliotis tuberculata) hemocyanin protein, with KLH andConcholepas concholepas hemocyanin protein being preferred.

Conjugation of GnRH or its analog to the mollusk hemocyanin protein ispreferably conducted using a cross-linking agent to allow a large numberof GnRH or analog molecules (i.e., 200 or more) to be coupled to asingle carrier protein molecule, effectively covering its outer surfacewith consistently aligned epitopes of the GnRH displaying the same basicconformation. To ensure this consistent alignment, the GnRH (or itsanalog) is coupled through its C-terminal end to the N-terminal end ofthe carrier protein through a bifunctional cross-linking agent. In aparticularly preferred embodiment, the GnRH/carrier conjugate may beshown by the formula:(X-A_(m)-B-L)_(n)-R  (I)wherein X is GnRH or a GnRH immunogenic analog, A is an optional aminoacid spacer such as Gly, m is an integer greater than or equal to 0, Bis an amino mercaptan, R is an intact immunogenic mollusk hemocyaninprotein, L is a bifunctional crosslinking agent effective forsimultaneously binding to the thiol of the mercaptan and to free aminemoieties of the immunogenic mollusk hemocyanin protein, and n is aninteger greater than or equal to about 200. A variety of aminomercaptans may be used, provided that it possesses a free amino moietyfor binding to the C-terminal end of X (or A if present) and a freethiol moiety for binding to the bifunctional crosslinking agent,although cysteine is preferred. Preferred bifunctional crosslinkingagents includesuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) orsulfo-SMCC (s-SMCC), either of which form a maleimide-activated carrierprotein. Other crosslinking agents suitable for conjugating the carrierprotein and GnRH through the thiol group of the amino mercaptan includebut are not limited to the organic solvent soluble agents Succinimidyl4-(p-maleimidophenyl)-butyrate (SMPB),-[(γ-Maleimidobutyryl)oxy]succinimide ester (GMBS),-Succinimidyl[4-iodoacetyl]-aminobenzoate (SIAB), andm-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS), or theircorresponding water soluble sulfonated forms sulfo-SMPB (s-SMPB),sulfo-GMBS (sGMBS), sulfo-SIAB (s-SIAB), and sulfo-MBS (s-MBS).

Preparation of the above-mentioned GnRH/mollusk hemocyanin proteinconjugate is preferably conducted under conditions of approximatelyneutral pH and high salt concentrations to prevent the disassociation ofthe protein into subunits, and thereby prevent mollusk protein epitopesfrom being exposed to the animal's immune system. Thus, the protein ispreferably dissolved in a buffer having an NaCl concentration greaterthan or equal to about 0.6 M, particularly about 0.9 M. Addition ofsucrose to the carrier protein solution is also preferred to reduce thedenaturation of the protein during freeze lyophilization processing andto allow the material to be rehydrated without precipitation. A detaileddescription of the conjugation procedure is provided in Example 2.

The GnRH or GnRH analog carrier conjugate is formulated with theadjuvant of the invention in the same manner described above for anyimmunogen of interest. However, when using compositions which include amollusk hemocyanin carrier protein, the vaccine composition willpreferably further include physiologically buffered saline with a highsalt concentration to prevent dissociation of the protein. The salt(NaCl) concentration of the vaccine composition is preferably greaterthan or equal to about 0.7 M and less than or equal to about 1.0 M, andthe pH of said vaccine composition is between about 7.0 and 8.0, with7.4 being preferred.

In this preferred embodiment, the invention provides a method forinducing anti-GnRH antibody by administering to a subject animal avaccine composition including the adjuvant containing killed cells of M.avium subsp. avium with the GnRH or GnRH analog conjugate. As noted, theproduction of the anti-GnRH antibodies effects the neutralization ofGnRH in the animal, thereby reducing LH and FSH blood levels andinhibiting the production of androgens and other steroids and sperm inthe testes of males, and inhibiting the production of progestogens andoestrogens and follicle maturation in the ovary of females. As aconsequence, the induction of anti-GnRH antibodies may be used foreffecting one or more treatments of animals, including the contraceptionof males and/or females, reducing aggressive behavior in male animals,chemical castration, control or prevention of estrus or heat, preventionof restlessness in animals prior to slaughter, reduction of boar taintin the meat of pigs raised for slaughter, and treatment of variousdiseases as noted hereinabove.

Accordingly, the GnRH or GnRH analog conjugate should be administered inan amount effective to induce one or more of these responses asdetermined by routine testing. For example, where the desired effect iscontraception, an “effective amount” is defined to mean those quantitieswhich will result in a significant reduction in fertility relative to anuntreated control animal. Infertility can be measured by methods knownin the art, e.g. by evaluation of spermatogenesis or ovulation, as wellas by statistical modeling of experimental animal data. Other indicatorsof infertility in males includes reduction of serum testosterone tocastration levels and involution of the testes. Similarly, where theultimate response is a reduction of aggressive behavior in male animals,an effective amount is defined to mean those quantities which willresult in a significant reduction in aggressive behavior of a test groupas compared to an untreated group. The actual effective amount will ofcourse vary with the specific GnRH or GnRH analog, the immunogeniccarrier and manner of conjugation, the target animal and its size, thedesired effect, and the treatment regimen (i.e., treatment with only asingle dose, or treatment with a first dose followed by a boost dose),and may be readily determined empirically by the practitioner skilled inthe art using an antigen dose response assay for each animal species.

Without being limited thereto, typical single shot doses of the GnRH orGnRH analog conjugate in the vaccine for the treatment of small animals(such as rodents, Norway rats, squirrels, rabbits, dogs, and domesticcats) will be between about 50 and 250 μg, for medium size animals (suchas pigs or deer) will be between about 400 and 800 μg, and for largeanimals (such as cattle, bison, horses, or elk) will be between about1,000 to 2,000 μg conjugate. The doses presented above are provided onlyas a guide for adult or full-size animals, and for animals encompassinga number of species or breeds, such as dogs, deer, or horses, theindicated dose is for the average or typical size animal, not“miniature” breeds or species. It is envisioned that doses for verylarge animals such as elephants would be considerably greater, andshould be determined empirically.

We have discovered that when the vaccine is formulated with the killedM. avium subsp. avium adjuvant of this invention, the above-describeddoses of GnRH or GnRH analog provide effective immunocontraception foran extended period of time, eliciting high anti-GnRH antibody titers forperiods of more than 1 year, after only a single dose or shot. Fortreatment programs utilizing two doses, a first primary dose and asecond boost dose, the doses described above for a single dose regimenshould be cut approximately in half for each dose.

The GnRH or GnRH analog containing vaccine of this invention areeffective for treatment of a broad spectrum of both wilds anddomesticated animals, ranging from pets, to large domestic or wildanimals, including mammals, birds, and reptiles. Without being limitedthereto, preferred animals which may be treated include porcine, bovine,equine, feline, canine, primates (including humans), Rodentia, Cervidae,and Pachydermata, and particularly domestic dogs, domestic cats, pigs(including captive or feral pigs), cattle, deer, horses, zoo animals,elephants, rodents (including rats, rabbits, and squirrels), andreptiles.

The vaccine may be administered to the subject animal by parenteralinjection (e.g., subcutaneous, intravenous, or intramuscular). Forimmunocontraceptive treatments, the vaccine should be administered priorto the desired onset of infertility to allow development of effectivelevels of anti-GnRH antibodies in the subject animal. Thus, the vaccinewill typically be injected at least about 3 months prior to the desiredtime of contraception.

The following examples are intended only to further illustrate theinvention and are not intended to limit the scope of the invention whichis defined by the claims.

EXAMPLE 1 Preparation of Adjuvant

Killed cells of Mycobacterium avium subsp. avium were obtained from thecommercial Johne's disease vaccine, a Mycobacterium avium bacterin(provided by Solvay Animal Health Products Inc., Mendota Heights, Minn.55120, Product No. 09149). Each vial contains a total of 25.5 mg killedcells of Mydobacterium avium (dry weight) in 0.5 ml of mineral oil.Vials are stored at refrigerator temperature, 36° to 45° F.

A mineral oil diluent is used for preparation of the adjuvant. In asterile 50 ml centrifuge tube, combine mineral oil (light white oil) andmannide monooleate (9:1 by weight) and vortex to mix. Store at roomtemperature in the sterile 50 ml vial.

A concentrated stock solution of the killed cells in mineral oil may beprepared for storage and subsequent preparation of adjuvant doses invaccine formulations. The Stock Solution is a 1/30 dilution of theoriginal Mycobacterium avium bacterin. Prepare in a sterile 50 mlcentrifuge tube. Add an entire vial (0.5 ml) of Mycobacterium aviumbacterin to 10 ml of the diluent. Rinse the Mycobacterium avium bacterinvial 2 times with 1 ml of diluent; each rinse is added to the 50 ml vialbringing the total volume to 12.5 ml, then add an additional 2.5 ml ofdiluent to the stock vial bringing the total volume to 15 ml Vortex tomix. Vials are stored at refrigerator temperature. The concentration ofkilled cells in the stock solution is 1.7 mg/ml.

The stock solution is diluted as necessary to prepare both the prime andthe boost dose adjuvants used in the vaccines. The primary dose adjuvantis a 1/150 dilution of original Mycobacterium avium bacterin. In asterile 50 ml centrifuge tube, make a ⅕ dilution of the stock solutionwith the diluent. Add 1 ml of the Stock Solution to 4.0 ml of thediluent. Vortex to mix. The concentration of killed cells in the primedose adjuvant is 340 μg/ml. Vials are stored at refrigeratortemperature.

The boost dose Adjuvant is a 1/300 dilution of the Mycobacterium aviumbacterin. In a sterile 50 ml centrifuge tube, make a 1/10 dilution ofthe stock solution with the diluent. Add 1 ml of the stock solution to9.0 ml of the diluent. Vortex to mix. The concentration of killed cellsin the prime dose adjuvant is 170 μg/ml. Vials are stored atrefrigerator temperature.

EXAMPLE 2 GnRH-KLH Vaccine Preparation

Preparation of MSB Buffer:

Add 7 tablets of Sigma Phosphate Buffered Saline (PBS) tablets to 200 mldistilled H₂O, to give a 0.07 M Phosphate buffer at pH 7.4 with 0.96 MNaCl. Add 5.6 gm Sucrose (41 mM Sucrose) to the PBS solution. For longterm stability the buffer is frozen. MSB is stable for about 30 days inrefrigerator.

Preparation of GnRH/KLH conjugate:

KLH carrier protein is first subjected to maleimide activation foraddition of sulfide binging groups thereto. 10 mg of the mollusk proteinKLH is dissolved in the MSB buffer, and 2 mg of sulfo-SMCC is added(Pierce Chemical Co.) with gentle mixing to dissolve. The mixture isallowed to react for 1 hour at room temperature with periodic mixing.After completion of the reaction, the maleimide-activated protein isimmediately purified by applying the reaction mixture to a desaltingcolumn (i.e., Sephadex G-25). The maleimide activated protein comes offon the void volume (first peak, fractions 4-6) as measured by absorbanceat 280 nm. There is a drop in absorbance after fraction 6, and a rise inabsorbance in fractions 7 or 8 as the excess sulfo-SMCC comes off.Excess cross-linker is removed in order to achieve good conjugation tothe hapten. At this point the maleimide activated KLH may be frozen orfreeze dried.

GnRH Hapten Conjugation:

Six mg of the GnRH-Gly-Cys hapten (containing a free SH on one end) isdissolved in 1 ml of H₂O. The dissolved hapten is added to the activatedKLH and allowed to react for 2 hours at room temperature and thenovernight in the refrigerator. The KLH-hapten conjugate is immediatelypurified by applying the reaction mixture to a desalting column.(Sephadex G-25). The conjugate protein should come off on the voidvolume (first peak, fractions 4-6) as measure by absorbance at 280 nm.There should be a drop in absorbance after fraction 6 and rise inabsorbance in fractions 7 or 8 as the excess hapten comes off. A smallamount of excess hapten does not cause problem in a prime dose but couldneutralize antibody in a boost dose if it is present in large excess.The GnRH/KLH conjugate may be frozen or lyophilized or used in thepresent form.

GnRH Vaccine Formulation:

Primary dose formulations of the vaccine are prepared by mixing equalportions (1:1 ratio) of GnRH-KLH conjugate (0.5 ml) with the prime doseadjuvant of Example 1 ( 1/150) (0.5 ml). The final vaccine dose shouldcontain approximately 170 μg of killed bacteria per 0.5 ml dose. TheGnRH conjugate must be added to the oil adjuvant (not oil to GnRH) in adrop wise manner while the oil is vortexed. This forms a milk likeemulsion. The emulsion is stiffened by passing through a 22 gauge needle3 times.

Boost dose formulations of the vaccine are prepared by mixing equalportions (1:1 ratio) of GnRH-KLH vaccine (0.5 ml) with the boost doseadjuvant of Example 1 ( 1/300) (0.5 ml). The final vaccine dose shouldcontain approximately 85 μg of killed bacteria per 0.5 ml dose. Again,the GnRH conjugate must be added to the oil adjuvant (not oil to GnRH)in a drop wise manner while the oil is vortexed. This forms a milk likeemulsion. The emulsion is stiffened by passing through a 22 gauge needle3 times.

EXAMPLE 3 Immunocontraception of Deer

The GnRH immunocontraceptive vaccine of Example 2 was used for theimmunocontraception of deer as either a two shot or single shot vaccine.

Two Shot Trial:

Deer were injected with a first, prime boost, followed by injection 1year later with a second, boost injection of the GnRH/adjuvant vaccinesof Example 1. The deer were injected with 1 ml of the vaccinecomposition. Titers of anti-GnRH antibodies and blood progesteronelevels were monitored over a two year period immediately prior to andfollowing treatment. The amount of the conjugate in each dose of thevaccine was 450 μg.

The results are shown in FIG. 1. Deer injected with a prime and boostvaccination of KLH-GnRH/Adjuvant in the breeding season of the firstyear of the trial have remained infertile through four consecutivebreeding seasons (four years) without a second or third season boostvaccine. Anti-GnRH antibody titers remained at 128,000 into the thirdyear, and dropped to 28,000 in the fourth year with deer remaininginfertile (FIG. 1). The two shot paradigm effectively contracepted deerfrom 2 to 4 years. Two out of the 3 deer tested were still infertileafter 4 years.

One Shot Trial with Female Deer:

Following the success observed after two mating seasons of the two shottrial, a second trial was commenced to determine efficacy using only asingle shot of the vaccine of Example 2. In July prior to the firstmating season of this single shot trial, 5 Penn State deer were injectedwith 1 ml of the vaccine composition. The dose of the GnRH conjugate wasincreased over that used in the two shot trial; the concentration of theGnRH concentration was 850 μg/ml. They were exposed to the bucks onNovember of that year. All five remained infertile for that year. FIG. 2represents a typical antibody response for the single shot deer. In the2nd year, 3 out of the 5 remained infertile, while two of the deer had asingle fawn indicating a partial protection.

Single Shot Trial with Male Deer:

One season after the single shot trial was initiated with female deer,male deer were subjected to a single shot trial. In July prior to thefirst mating season of this trial, 5 male deer were injected with asingle shot GnRH using the same amounts and concentrations of vaccine asin the single shot trial with the females (1 ml dose containing 850 μgof conjugate). In the November bleed, the testosterone levels of all 5deer were down to the level of sexually immature deer, and their antlershad prematurely dropped off. Therefore the single shot regimen waseffective in shutting the sexual activity of all male and female deerfor at least one year (Table 1).

EXAMPLE 4 Immunocontraception of Pigs

The GnRH immunocontraceptive vaccine of Example 2 was used for theimmunocontraception of pigs as a single shot vaccine.

The GnRH/adjuvant vaccine was tested in fifty 5 month old gilts. Pigs ofGroup 1 (n=10) were sham injected with the adjuvant only, while pigs ofGroup 2 (n=10) were given 2 oral doses of GnRH on mixed nut shells,Group 3 (n=10) were given a single dose containing 800 μg of GnRHconjugate, Group 4 (n=10) were given a single dose of 1600 μg of GnRHconjugate, and Group 5 (n=10) were given 2 doses of 400 μg of GnRHconjugate 30 days apart. At eight months of age or 3 months after thecontraceptive vaccine was given the gilts were checked for heat byteasing with a boar. The results which are dose relation are shown inFIG. 3. The 2 dose trial was the most effective giving a 100%contraceptive effect. However, the high single dose gave 90%contraceptive effect response.

As seen in FIG. 4 the heat cycles and pregnancy observed in the 8 monthsold female gild decreased as the GnRH antibody titer increased.

It is understood that the foregoing detailed description is given merelyby way of illustration and that modifications and variations may be madetherein without departing from the spirit and scope of the invention.TABLE 1 White Tailed Bucks Single Shot Anti-GnRH Testosterone (nq/dl)Testis (mm) Antlers Control 0 ± 0 477 ± 172 73 × 43 hardened One Shot48K ± 23K 4 ± 6 44 × 28 velvet or regimen shed

1-10. (canceled)
 11. In a method for vaccinating an animal against animmunogen of interest which comprises administering a vaccinecomposition to said animal which said vaccine composition comprises animmunologically effective amount of said immunogen in combination withan adjuvant effective to enhance the immune response in an animal tosaid immunogen, wherein the improvement comprises said adjuvantcomprising mineral oil and killed cells of Mycobacterium aviumsubspecies avium, the concentration of said killed cells ofMycobacterium avium being greater than or equal to about 50 μg per mland less than or equal to about 400 μg per ml, measured as the dryweight of said killed cells per ml of said vaccine composition, andfurther wherein the amount of said killed cells of Mycobacterium aviumin said vaccine composition is not sufficient to elicit a Tcell-mediated delayed hypersensitivity response to M. avium by saidanimal if said adjuvant was administered alone, without said immunogen.12. The method of claim 11 wherein the amount of said killed cells ofMycobacterium avium subspecies avium in said vaccine composition is lessthan or equal to about 400 μg, measured as the dry weight of said killedcells.
 13. The method of claim 12 wherein the amount of said killedcells of Mycobacterium avium subspecies avium in said vaccinecomposition is less than or equal to about 200 μg, measured as the dryweight of said killed cells.
 14. The method of claim 11 wherein saidanimal is selected from the group consisting of porcine, bovine, equine,feline, canine, primates, Rodentia, Cervidae, and Pachydermata.
 15. Themethod of claim 11 wherein said animal is selected from the groupconsisting of domestic dogs, domestic cats, pigs, cattle, deer, horses,zoo animals, elephants, rodents, and reptiles.
 16. The method of claim11 wherein said adjuvant further comprises a surfactant
 17. The methodof claim 11 wherein said immunogen is selected from the group consistingof GnRH or a GnRH immunogenic analog conjugated to a carrier protein,and porcine zona pellucida.
 18. The method of claim 17 wherein saidimmunologically effective amount comprises an amount effective forinducing immunocontraception of said animal.
 19. The method of claim 18wherein said immunologically effective amount comprises an amounteffective for inducing immunocontraception of said animal in a singledose.
 20. The method of claim 17 wherein said immunogen is mammalianGnRH or a mammalian GnRH immunogenic analog conjugated to a carrierprotein.
 21. The method of claim 11 wherein said immunogen comprisesGnRH or a GnRH immunogenic analog conjugated to KLH carrier protein, andsaid GnRH or GnRH immunogenic analog is conjugated to said KLH carrierprotein through the d-terminal end of said GnRH or GnRH immunogenicanalog.
 22. The method of claim 21 wherein said vaccine compositionfurther comprises physiologically buffered saline, and further whereinthe salt concentration of said vaccine composition is greater than orequal to about 0.7 M and less than or equal to about 1.0 M, and the pHof said vaccine composition is between about 7.0 and 8.0.
 23. The methodof claim 11 wherein said vaccine composition is administered byparenteral injection. 24-29. (canceled)